DE1670016C3 - Process for the production of 5'-ribunucleotides by selective phosphorylation - Google Patents

Description

3 4

The use of smaller amounts of salts of the trated residue was mixed with methanol, and

organic amine with an inorganic acid as the resulting mixture was gradually cooled,

the specified amounts lead to a decrease - whereby beautiful, needle-shaped crystals crystallize

the rate of phosphorylation.

reaction of the nucleoside and to an amplified 5 The crystals were filtered off and in vacuo

Formation of by-products. dried, 5.1 g of pure disodium 5'-gu-

As most salts of organic amines with anylate (purity 95.6%, water 3.2%).
inorganic acids are hygroscopic, it is dated

procedural point of view from an advantageous, Example 2
Amines and inorganic acid which io in the reaction

organic solvents used to form Example 1 was repeated with the difference,

of the desired salts in this solvent amount to 2.75 ml (20 ml mol) of pyrophosphoryl chloride

Separately, instead of using the preformed salts in place of phosphorus oxychloride. the

use. Conversion into guai> osine phosphates and into 5'-guanyl

The consumption acid in the process according to the invention was 98 and 93.1%, respectively.
bare amines are primary, secondary and tertiary

Amines, while inorganic acids such. B. B eis ρ ie 1 3
Hydrochloric acid, nitric acid, sulfuric acid or chloric acid can be used. To 15 ml of dichloromethane were added 3 ml (33 mmol)

It is to be regarded as surprising that in the case of *> phosphorus oxychloride and 7.3 g (46.2 mmol) «-picoline-

Phosphorylation of unprotected ribonucleoside given hydrobromide. The resulting mixture

using phosphorus oxychloride, phosphorus bromide was ^{cooled to 4 to 6 0} C and with stirring

or pyrophosphoryl chloride added the selectivity of the phos- 4.2 g (14.8 mmol) guanosine. After 5 hours

Phorylation of the primary hydroxyl group of the Ri- reaction was the reaction mixture in 600 ml

bonucleosids can be enhanced when the reac- as ice water is poured. The conversion into guanosine

tion in the presence of one of the aforementioned polar phosphates or 5'-guanylic acid was 94.5 and 85.2%, respectively.

organic solvent, which is a salt of a. The mixture was left at 0 to 5 ^° C. for about 2 hours

organic amine with an inorganic acid. The resulting aqueous solution was with

contains, is carried out. Sodium hydroxide adjusted to a pH of 3 and

A high conversion rate and a high 30 are then mixed with 120 ml of methanol. They left the However, the selectivity of the reaction will only then stand the solution at about 5 ° C. overnight. Who disentangles, when the reagents stood in the specified precipitate was filtered off and poured into 50 ml Quantities are used. dissolved in warm water, whereupon the solution in the

The process according to the invention is treated with methanol in the manner described above

explained in more detail in the following examples. Was 35. The resulting precipitate was with

Dissolve 200 ml of water and, after the pH of the

Example 1 solution was adjusted to 7.0 to 8.0, the

Solution run through a column containing 50 ml

2.75 ml (30.3 mmol) of anion exchanger (Cl form) were added to 15 ml of acetonitrile. The co-

Phosphorus oxychloride and 8.3 g (73.2 mmol) of pyridine 40 lonne were then first washed with water

Given hydrochloride. The resulting mixture, that and then with dilute aqueous hydrochloric acid solution

was kept at 0 to 2 ° C, was eluted with 4.2 g. Then, as in Example 1, further

(14.8 mmol) of guanosine were added. The reaction driving. 4.7 g of Dinatnum 5'-guanylate were obtained

mixture, which was initially present as a suspension, was added (purity 96.1%, water 2.5%).
progressive reaction gradually into a full 45

constantly clear solution over. After 4 hours of reaction, Example 4
the reaction mixture was mixed with 150 ml of ice water

added to give an aqueous solution. Example '. *: ■■■ <& repeated with the deviation,

An analysis of the resulting aqueous solution that on Sfe "c ν - · ι-> acetonitrile used nitromethane

made sure the conversion to guanosine turned 50. The conversion into guanosine phosphates and

phosphate and 5'-guanyl acid was 97.6 and 92%, respectively, in 5'-guanylic acid was 98.2 and 92.3%, respectively. Man

was done. received 5.05 g of disodium 5'-guanylate (purity 94.8%,

After the aqueous solution for 2 hours with water 3.7%).

was kept about 5 ° C, it was carried out by a method described in Example 5
150 ml of granulated activated carbon charged acid 55

sent to adsorb the guanylic acid. Example 1 was repeated with the difference

The column was then filled with about 600 ml of water so that dioxane was used instead of acetonitrile,

wash and then with 0, ln-aqueous sodium hydro- The conversion into guanosine phosphates and 5'-Gu-

xyd solution eluted. The eluate after the monoylic acid was 89.4 and 83.8%, respectively. One received

Adjustment of its pH to less than 8.0 by means of a 60 4.7 g disodium 5'-guanylate (purity 96.8%, water

Column sent with an anion exchanger- 1.8%).

Resin (Cl form) was filled to ad- examples
sort by. Then the column was initially with

Water and then washed with a 0.03n- Example 1 was repeated with the difference,

aqueous solution of hydrogen chloride eluted. In 65 that a solvent mixture consisting of 10 ml

the eluate was adjusted to pH 7.6 acetonitrile with sodium hydroxide and 5 ml propionitrile, instead of acetonitrile

set. The eluate was used under reduced tonitrile alone. The conversion to

Pressure concentrated up to a syrup. The concen- trated guanosine phosphate and in 5'-guanylic acid was

97.3 and 92.4%, respectively. 4.8 g of disodium Example 10 were obtained
5'-guanyate (purity 96.3%, water 2.1%).

. . Example 1 was repeated with the difference

ö e ι s ρ ι e I 7 _{that 3ß5 g (15 mMo} r) undine instead of guanosine

To 15 ml of acetonitrile containing 2.7 g (74 mmol) of 5 was used. The conversion into uridine

Hydrogen chloride, 2.5 ml (27.5 mmol) of phosphates and 5'-uridylic acid were 98 _t 2 and

phosphorus oxychloride and 5.2 ml (64.4 mmol) pyridine 94.0%. 4.6 g of sodium 5'-uridylate (purity

given. The resulting mixture was 97.8%, water 1.1%).

with stirring at 0 to 5 ^C 4 g (14.9 mmol) inosine example 11

given. After 2 hours of reaction, the io

150 ml of ice water are added to the reaction mixture. Example 7 was repeated with the difference

The paper electrophoresis of the resulting aqueous that 5.5 g (68 mmol) of hydrogen bromide instead of

Solution revealed that the conversion to inosine phosphate was using hydrogen chloride. One received

and in 5'-inosinic acid was 99% and 95%, respectively. 5.3 g disodium 5'-inosinate (purity 99.0%, water

Then wu.de continue as in example 1. 15 0.8%).

5.5 g of disodium 5'-inosinate (purity _n _ _{; c} ".- _ 1 η

98.4%, water 1.0%). Examples

Example 1 was repeated with the difference

Example 8 that 10 g (72.5 mmol) of triethylamine hydrochloride

in place of pyridine hydrochloride were used.

15 ml of acetonitrile were mixed with 2.7 ml (29.7 mmol). The conversion into guanosine phosphates and 5'-Gu-

Phosphorus oxychloride and 7.4 g (65.2 mmol) of pyridianylic acid were 94.7 and 86.5%, respectively. One received

nium chloride added. This mixture was added to 0 to 4.9 g of disodium 5'-guanylate (purity 96.3%, water

Maintained 2 ° C and with 4 g (15 mmol) adenosine below 2.1%).

Stirring added. After 2.5 hours of reaction, 35 ice cream was 13
the reaction mixture mixed with 200 ml of ice water.

The conversion into adenosine phosphate and 5'-adenyl- Example 1 was repeated with the difference

acidity was 93.7 and 87.9%, respectively. The resulting aqueous that 11 g (40.5 mmol) of morpholine-sulfuric acid salt

Solution was treated with activated charcoal in the way that pyridine hydrochloride was used in place of

as described in Example 1. Then she turned 30 The conversion into guanosine phosphates and into

sent through a column containing an anion 5'-guanylic acid was 95.3 and 85.5%, respectively. One received

exchanger (Cl form) was filled. The column 4.7 g of sodium 5'-guanylate (purity 95.7%, water

was first washed with water and an- 2.8%).

finally eluted with 0.05N aqueous hydrochloric acid, Example 14

only adenic acid was eluted. 35

The eluate was concentrated at low temperature - Example 1 was repeated with the difference that

trated and the concentrated residue was with that instead of guanosine 4.3 g (15 mmol) of xanthosine

Methanol was added and the resulting mixture was allowed to be used. The conversion into xanthosine

to stand in a refrigerator. phosphate and 5'-xanthylic acid was 90.1 and

Beautiful crystals could be filtered off as precipitate 40-85.1%. 4.7 g of disodium 5'-xanthylate were obtained

will. After drying, 4.5 g (purity 94.8%, water 3.5%) were obtained.

5'-adenylic acid (purity 99.1 · / ·, water 0.5%). .

Example 9 Example 10 was repeated with the difference

Example 1 was repeated with the difference that 13 g (45.3 mmol) of phosphorus oxybromide were used instead that 3.65 g (15 mmol) of cytidine were used in place of guanosine of phosphorus oxychloride. the were used. The conversion to cytidine- Conversion to uridine phosphates and 5'-uridyl phosphates and 5'-cytidylic acid was 95.6 and acid was 99.2 and 96.4%, respectively. 4.8 g were obtained 88.8%. Sodium 5'-uridylate (purity 99.2%, water 0.1%).

Claims (3)

font 1428 186 are solutions of nucleosides _Claim : after adding metaboric acid to protect the 2 ', 3'-hydroxyl groups heated and then with Phos- Process for the production of 5'-RibonucIeophoroxychlorid on the 5'-Hydroxygruppe phosphorytiden by selective phosphorylation of the ent- S liert. Finally, the reaction solution is hydrolyzed after appropriate ribonucleoside with the help of a phosphorus removal of unreacted phosphorus oxychloride by phosphorus halogen compound and subsequent hydro- addition of ethanol,
lysis, characterized in that carrying out these various reactions the ribonucleoside in more than 1 liter, however, requires a lot of work, since the reaction conditions of the desired tril, propionitrile, nitromethane, nitroethane, tetra reactions must be set based on one mole of the ribonucleoside, acetonio,
hydrofuran, dioxane, dichloromethane, chloroethane, attempts have already been made to remove unprotected nucleosides Dimethyl sulfoxide, dimethylformamide, acetone, to be phosphorylated selectively (G u 11 a η d and H ο b-methyl ethyl ketone or their mixtures as day, J. Chem. Soc, 1940, p. 746; Barker and solvents with phosphorus oxybromide, chloride 15 Foil , J. Chem. Soc, 1959, p. 3798 and T e η er, or pyrophosphoryl chloride in an amount from J. Chem. Soc, 1961, [Vol. 83], p. 159). In these over 1 mol up to 5.0 mol per liter of the known solution process, however, the conversion and the selectivity of the reaction by means of an organic salt in the presence of an amine with an inorganic acid are so unsatisfactorily low that they are not practical Have gained importance. Amount of about 0.5 to 2.5 MoI per mole of the ao It is therefore an object of the invention to provide a method of phosphorus halogen compound at -30 to +30 ⁰ C for the preparation of 5'-phosphorylated RibonucIeotiden accessible from 0.5 to 10 hours, and then to make, in which the selective phosphorylation siert the reaction mixture in the usual way hydrolyzing the primary hydroxyl group of unprotected. Ribonucleosides with good selectivity and yield 25 can be carried out. The invention therefore relates to a method for the production of 5'-ribonucleotides by selective Phosphorylation of the corresponding ribonucleoside with the aid of a phosphorus halogen compound and The invention relates to a method for the production of subsequent hydrolysis, which is characterized by of 5'-ribonucleoside by selective phosphory- is that the ribonucleoside in more than 1 liter, lation of ribonucleosides. According to the invention, based on 1 mol of the ribonucleoside, acetonitrile, economically produced 5'-ribonucleotide propionitrile. Nitromethane, nitroethane, tetrahydro- are suitable as chemical reagents or as seasoning furan, dioxane, dichloromethane, chloroethane, dimethyl and seasoning components. 35 sulfoxide, dimethylformamide, acetone, methylethyl For phosphorylation of a primary hydroxy ketone or mixtures thereof as a solvent with group of a nucleoside, it has so far mostly been used for phosphorus oxybromide, chloride or pyrophosphoryl held necessary, the secondary hydroxyl groups chloride in an amount of over 1 up to 5.0 mol of the nucleoside with the aid of suitable groups per liter of the solvent in the presence of a salt protection. 40 of an organic amine with an inorganic The following three reaction stages must be acid in an amount of about 0.5 to 2.5 mol per mol are conducted: the phosphorus halogen compound at -30 to +30 ⁰ C Production of 2 ', 3'-O-protected ribonu- phosphorylated for 0.5 to 10 hours and then the cleosid reaction mixture hydrolyzed in the usual way. 2. Production of 2 \ 3'-O-protected Ribonu- ^4S When the process according to the invention is carried out in practice, the process according to the specified 3. Splitting off of the protective groups in the 2 ', 3' position. Temperature maintained solvent phosphoroxy chlond, bromide or pyrophosphoryl chloride as well An example of this known process is the salts of organic amines with the inorganic French patent specification 1 417 811, according to which as 50 acids are added with stirring, whereupon the ent-protecting group is the isopropylidene group used to form an unprotected ribonu, which in the last Process stage given by heating cleosid and stirring. Is removed again by mixing in acidic solution. of the resulting reaction mixture with water It is known that these three reactions are carried out in one step without residual phosphorus oxyhalide or bromide, isolation of intermediates, or pyrophosphoryl chloride and the resulting supply (compare, for example, J. Am. Chem. Soc, Vol. 83, Phosphordihalogenierte Nucleosid The aqueous solution resulting from p. 3640 [1961] and the French patents is used in that for 1,434,511 and 1,428,186). Treated according to the customary French purification of nucleotides, patent specification 1,434,511, first the T- and, in order to obtain the desired 5'-ribonucleotide, the 3'-hydroxyl groups of the nucleoside in the presence of 60 The solvents mentioned are polar solutions of phosphorus oxychloride in a solvent in medium. Non-polar solvents are converted to the isopropylidene group; then the method according to the invention is not suitable. Pyridine is also added to the reaction mixture and, without first isolating solvents such as carboxylic acids, amines and the 2 ', 3'-O-isopropylidene nucleoside, alcohols are unsuitable because they phosphorylate the 5'-hydroxy group as a side reaction and then ionize Formation of by-products result in the hydrolysis of the protective groups by heating to a greater extent. The specified acidic solution medium carried out. Means can be used alone or in a mixture According to the method of the French patent.